Creating a deletion by PCR splicing

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Author Dr. Alexei Gratchev

This approach can be used to delete a fragment of any lenth or to introduce point mutations into a DNA seqence.

To delete a desired fragment from existing DNA fragment all you need is a pair of primers flanking the region where the deletion will be made (primers 1 and 4), 2 compltementary primers comprising a region of -15 bp to +15 bp related to the junction point (primers 2 and 3) and a high fidelity polymerase (a mix of Taq and Pfu for example). The procedure is shown on the picture. It is always helpful to create expected sequence using any sequence editing software first and then choose primers using this "virtual" construct. Following is important for carrying out the experiment:

  • If you use the plasmid as a template use about 500 ng. In this case you can make only 20 cycles to have a good product.
  • Primers 1 and 4 can be any, primers 2 and 3 should be about 30 bases (15 per flank), but you can anyway use annealing at 55°C.
  • Usually no purification of the first stage products is needed, just dilute them 1:100 to reduce the amount of the primers 2 and 3 which you don't need any more.
  • If the deletion is so short that you cant separate deleted and wt products on the gel, you have to gel-purify the products after the 1-st step.
  • Procedure for the point mutation is the same, but selecte primers 2 and 3 related to the mutation point and introduce the mutation in the primer sequence.

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\METHODS\Mutagenesis\PCR splicing